Biofuel''''s Engineering Process Technology Part 4 pot

Nanotech Biofuels and Fuel Additives

111
9. Public concerns over nanotechnology: security, health and the
environment
As with all new technologies, there may be cause to concern about impacts, such as on
security, health and the environment. Nanotechnologies have been the subject of many
assessments seeking to anticipate possible consequences of their deployment, to humans
and to the environment. For instance, the Woodrow Wilson Center carried out a
Nanotechnology project [25] from 2005. The project managers said that “manipulating
materials at the atomic level can have astronomic repercussions, both positive and negative.
The problem is no one really knows exactly what these effects may be.” This was the
motivation for the Project on Emerging Nanotechnology at the Woodrow Wilson Center.
Another initiative came from the International Risk Governance Council – IRGC’s
Nanotechnology project [26]. Two expert workshops were held. The first in May 2005
focused on how to frame nanotechnology, its risks and its benefits. A distinction was made
between the nanotechnologies of the so-called Frame One (passive or classical technology
assessment) and Frame Two (active or the social desirability of innovation). The second, in
January 2006, concentrated on identifying gaps in nanotechnology risk governance and
developing recommendations for improved risk governance.
A symposium on the subject took place in Zurich in July 2006. A presentation by Ortwin
Renn[27] discussed the policy implications of Frame One, referred to in Fig. 4. The fact is
that “most people have no clear associations when it comes to nanotech. They expect
economic benefits but no revolutionary technological breakthroughs. Risks are often not
explicitly mentioned but there is a concern for unforeseen side effects. There is a latent
concern about industry, science and politics building a coalition against public interest. And
one negative incident could have a major negative impact on public attitudes.”


Fig. 4. Frames of reference of nanotechnology generations

Biofuel's Engineering Process Technology

112
The IRGC’s Nanotechnology project concluded[28], among other things, that
“communication about nanotechnology’s benefits and risks should reflect the distinction
between passive and active nano-materials and products, stressing that different approaches
to managing risks are required for each. Care should also be taken to ensure that potential
societal concerns about the possible impacts of Frame Two active nano-materials do not
have the effect of unnecessarily increasing anxiety regarding Frame One products using
only passive nanostructures.” This is further expounded by Renn [29] as follows: “Frame
One passive nanostructures are found, for example, in easy-to-clean surfaces, paints or in
cosmetics. Frame Two refers to active nanostructures and molecular systems which could be
able to interact actively or could be understood as evolutionary biosystems which change
their properties in an autonomous process.”
In reality, nanotechnologies are already facing challenges. Man-made nano-materials have
been banned by the UK Soil Association from all its certified organic products. The 2008
annual report of the Soil Association of the UK contains the following statement [30]: “The
Soil Association published the world’s first standards banning nanotechnology. The risks of
nanotechnology are still largely unknown, untested and unpredictable. Initial scientific
studies show negative effects on living organisms, and three years ago scientists warned the
Government that the release of nanoparticles should be ‘avoided as far as possible’. There
are many parallels with GM in the way nanotechnology is developing, particularly because
commercial opportunities have run ahead of scientific understanding and regulatory
control. What’s more, while nano-substances are being rapidly introduced to the market,
there is no official assessment process or labeling of the products – which is even worse than
GM.
Health and beauty products that use nanoparticles are of concern for their potential toxicity
if they get under the skin. Similar concerns exist regarding food and textiles. Definitely,
more studies about health and environmental impacts are needed, to alleviate public

concerns.
On the other hand, there is so much potential for nanotechnologies to do good, that Frame
One and Two assessments should proceed as new applications evolve, including for
instance more effective delivery of drugs to fight human and animal disease.
Fig. 5 showing a RNA nano-particle created by Peixuan Guo of Purdue University,
illustrates the point. Strands are spliced together from two kinds of RNA – a scaffold and a
hunter to find cancer cells. This nano-structure has proven effective against cancer growth in
living mice as well as lab-grown human nasopharyngeal carcinoma and breast cancer cells.
10. Conclusions
Increasing demand for energy services in the decades ahead will require an expanding
supply of liquid fuels, despite efforts at improving energy efficiency and diversification of
energy systems, including growing use of electricity in transportation. Biofuels have a key
role to play in this scenario. However, the future supply of biofuels must be of such a scale
that non-food feedstocks and new technologies are intensively employed. Nanotechnologies
are primary candidates to play a prominent role in this energy future. They will help bring
to markets liquid biofuels, including renewable hydrocarbons, from algae, carbohydrates,
fatty esters and biogas. Nanotechnologies will also play a role in augmenting the efficiency
of using current and future liquid fuels, especially biofuels, by providing improved


Nanotech Biofuels and Fuel Additives

113

Fig. 5. RNA nano-particle created by Peixuan Guo, Purdue University [31]
combustion of nanodroplets. While there are risks in each and every new technology, the
world today is much better equipped to assess risks and act accordingly, that it seems
possible to advance nanotechnologies applied to biofuels, without jeopardizing security,
public health or the environment. But, the reach of nanotechnologies is vast and goes much
beyond biofuels and offer hopes in so many areas, including importantly, human health.

11. References
[1] Trindade, Sergio C. (2010). Refining will definitely survive. Pipeline Magazine, 29 August
2010
[2] Trindade, Sergio C. (2010). Renewable Energy Perspective – a profitable pathway from oil,
Exploration and Processing, Fall 2010 [8-9], Sep.
[3] Trindade, Sergio C. (2010). International Biofuels Trade: Issues and Options. International
Biofuels Conference_São Paulo, 26-28 May.
[4] Santana, G. and S. Quirk (2009). Growing Green: An In-Depth Look at the Emerging Algae
Industry, Greener Dawn Research, 22 July, 16p.
[5] A Sustainable Biofuels Consensus (2008). Statement from a conference hosted by the
Rockefeller Foundation Bellagio Study and Conference Center, Bellagio, Italy, 24-28
March 2008
[6] www.defra.gsi.gov.uk (2007), In: F.O. Lichts’s World Ethanol and Biofuels Report, Vol. 4,
No. 16, p.365 and Vol. 4, No. 17, p.391, Turnbridge Wells, U.K.: F.O. Licht, 2006.
[7] Carvalho da Silva, Flávio; Paulo Roberto da Costa Brum and Taís Neno dos Santos
(2005). Nanotechnology/Nanoscience Knowledge Managament emphasizing
nanostructured polymers. Presentation, School of Chemistry, UFRJ, Brazil.

Biofuel's Engineering Process Technology

114
[8] Borschiver, Suzana; Maria José O. C. Guimarães, Taís N. dos Santos, Flávio C. da Silva,
Paulo Roberto C. Brum (2005). Patenteamento em Nanotecnologia: Estudo do Setor de
Materiais Poliméricos Nanoestruturados., Polímeros: Ciência e Tecnologia, vol. 15, n°
4, p. 245-248.
[9] />of-algal-bio-fuel-production, April 23, 2009
[10]
[11] />biofuel-oil-without-harming-algae
[12] />biofuel-oil-without-harming-algae
[13] />facilities

[14]
Nov. 4, 2010
[15]
[16]
[17] Nanotechnology Used In Biofuel Process to Save Money, Environment Science Daily (Oct.
10, 2009)
[18]
[19] (U.S. Department of Energy. Berkeley Lab Helios Project. (n.d.) Helios Solar Energy
Research Center. Goals and challenges. Retrieved December 10, 2009 from

[20]
[21] Cleaner diesel engines – pouring water on troubled oils, The Economist, June 3
rd
, 2010, p.86

[22]
[23] Wulff, Pascal; Lada Bemert, Sandra Engelskirchen and Reinhard Strey (2008). Water-
biofuel microemulsions. Institute for Physical Chemistry, University of Cologne.
/>L_MICROEMULSIONS.pdf

[24] Strey, R. et al (2007). Microemulsions and use thereof as a fuel. US Patent Application
2007/028507 , Feb. 8.
[25]
[26] html
[27] />Ortwin_Renn_Nanotechnology_Frame_1_Policy_Implications_.pdf&name=Ortwin
+Renn&cat=document&showads=1
[28]
[29] p.14
[30]
&taid=303, p.22

[31]
6
Bioresources for Third-Generation Biofuels
Rafael Picazo-Espinosa, Jesús González-López and Maximino Manzanera
University of Granada
Spain
1. Introduction
Modern societies’ welfare relies greatly on fossil fuels. The current energy model, based on
the extensive utilization of fossil fuels, is affected by economic and environmental problems.
The United States Department of Energy 2009 report estimates that, within the next two
decades, global energy consumption will double (Conti, 2009). On the other hand, the
European Commission 2009 report indicates that the management of climate change
problems in Europe, since 2000, has been globally unfavourable. Nevertheless, there are
some positive signs, such as the 1.4% reduction in 2007 of CO
2
emissions with respect to the
figures obtained from 2000 to 2004 in the European Union of Fifteen (E-15). However,
considering the 27 European states (E-27), and paying attention to the consumption and
production of renewable energy and biofuels, the reduction in emissions has not fulfilled the
European Union objectives. Among the motives of this negative evaluation, the fall in the
companies’ productivity, increased transport and industry emissions and the reduction in
research and development areas can be cited (Radermacher, 2009). First- and second-
generation biofuels could ameliorate or solve the associated fossil fuel depletion problems,
although their recent implantation has raised some doubts. The main problems associated
with biofuels are the food vs. fuel controversy; the agricultural and forestry land usage and
the actual sustainability of biofuels’ production. Third-generation biofuels, based on the
microbiological processing of agricultural, urban and industrial residues, could be a possible
solution. However, several technical problems must be solved to make them economically
viable and easily affordable for the industry (Robles-Medina et al., 2009).
2. First-generation biofuels

The parallel progression in energy demands over depleting oil reserves and rising
greenhouse gas emissions entails a high risk of severe impacts on biodiversity, humankind
food security and welfare. Thus, a new energy model is needed, based on greener and
renewable energy sources, and cleaner as well as more sustainable fuel technology (Fortman
et al., 2008; Jegannathan et al., 2009).
2.1 Biogas, syngas, vegetable oils blends and Fischer Tropsch liquids
The first response of heavy industry to the current energy and environmental problems
includes some old systems, such as syngas and Fischer Tropsch liquids. Current advances in
technology and engineering could bring new opportunities to these classical chemistry and
biochemistry solutions, associated with fuel shortage situations such as the Arab oil
embargo of the 1970s, or the Second World War. Some of these will be detailed below.

Biofuel's Engineering Process Technology

116
2.1.1 Biogas
Biogas is an attractive source of energy primarily because it is renewable and enables the
recycling of organic waste. The production of biogas from manure can help to manage the
problems associated with this residue, contributing to the reduction of the greenhouse gas
methane. Besides, biomethanation is not only useful for energy production, but also for
cleaning up solid waste in urban areas. Compared with bioethanol from wheat and biodiesel
from rapeseed, biogas production based on energy crops could generate about twice the net
energy yield per hectare per year. Furthermore, biogas could be produced from the by-
products generated by the current bioethanol and biodiesel industries (Jegannathan et al.,
2009).
Biogas production is based on bacterial methanogenesis in the absence of air of organic
matter in a water solution. The process occurs in three steps. The first, hydrolysis, is carried
out by strict anaerobes such as Bacteroides or Clostridia, and facultative anaerobes such as
Streptococci. It involves the enzymatic transformation of insoluble organic material and
higher molecular mass compounds such as lipids, polysaccharides, proteins, nucleic acids

etc. into soluble organic materials — energy and cell carbon sources such as
monosaccharides and amino acids, among others. In the second step, acidogenesis, other
types of microorganisms ferment the mentioned products to acetic acid, hydrogen, carbon
dioxide and other lower weight simple volatile organic acids, such as propionic and butyric
acid, which are converted to acetic acid. Finally, organic acids, hydrogen and carbon dioxide
are converted into a mixture of methane and carbon dioxide by the methanogenic bacteria
such as Methanosarcina spp. or Methanothrix spp. (consuming acetate), as well as
microorganisms such as Methanobacterium sp. and Methanococcus sp., or others that consume
hydrogen and formate to yield methane (Jegannathan et al., 2009).
In spite of its attractions, biogas has only been used in rural areas of developing countries
and has received investment from governmental and non-profit organizations. The absence
of private investment is due to some technical limitations that hamper its economic viability.
The process is relatively slow and unstable, and requires large volumes of digester. The
decrease in gas generation during the winter season is a serious problem, and can lead to the
clogging of the reactor. Other causes for the reduction in gas production are pH and
temperature variations, so the loading rate and solid concentration have to be continuously
maintained (Jegannathan et al., 2009).
2.1.2 Syngas, biosyngas and Fischer-Tropsch derivatives
Synthetic gas, known as syngas, is a mixture of H
2
, CO and CO
2
in different proportions.
Traditionally, syngas was produced through gasification of coal at high temperatures, but it
can also be produced by methane reformation (submitting the methane to a high
temperature water steam stream, or hydrocracking) or by gasification of biomass. In the
latter case, the obtained gas is called biosyngas. Syngas and biosyngas can be used directly
as fuel, but they also can serve as precursors for other fuels, such as hydrogen, obtained by
the compression of carbon monoxide and dioxide. Also, by Fischer-Tropsch synthesis (FTS),
short and long chain hydrocarbons can be obtained from the aforementioned H

2
, CO and
CO
2
mixture (Srinivas et al., 2007). Fischer-Tropsch synthesis was discovered in the first half
of the twentieth century and developed for large-scale production during the Second World
War. It is based on the polymerization, through successive stages, of H
2
with CO and CO
2
,
yielding linear hydrocarbons. Iron, cobalt or ruthenium can be used as catalysts (Huber et
al., 2006). FTS can be developed at high or low temperature. The high temperature FTS is

Bioresources for Third-Generation Biofuels

117
performed at 330–350ºC yielding mostly short-chain hydrocarbons (gasolines) and light
olefins in a fluidized-bed reactor. On the other hand, low temperature FTS develops at 220–
250ºC in a slurry bubble column reactor, and waxes and long-chain hydrocarbons are
obtained (Bludowsky & Agar, 2009). As FTS is an extremely exothermic reaction, it can be
coupled with biomass gasification. However, FTS has some drawbacks, such as the fact that
complex mixtures of different chain lengths are always obtained. Thus, FTS products have
to be separated prior to subsequent processes (Huber et al., 2006).
2.1.3 Vegetable oil blends
The direct usage of crude or filtered vegetable oils for diesel engine fuel is possible by
blending them with conventional diesel fuels in a suitable ratio. These blends are easy to
obtain and keep stable for short-term use. But vegetable oils present high viscosity, acid
contamination and free fatty acids that lead to gum formation by oxidation, polymerization
and carbon deposition (Ranganathan, 2008). Thus, the long-term utilization of vegetable oils

for fuel leads to filter clogging, nozzle blockage and deposits in the combustion chamber
(Sidibé et al., 2010). Alongside the long-term problems in injection systems, filters and
combustion chamber, doubts about the sustainability of using crude vegetable oil for fuels
have to be considered. Vegetable oils are expensive, and their direct use in engines or as
feedstock to produce petro-diesel substitutes would encounter the same economic and
environmental problems that affect the conventional biodiesel and bioethanol industries
(UNCTAD, 2010).
A more interesting solution is the usage of waste cooking oil (WCO; also called waste frying
oil, WFO). Waste cooking oil is widely produced, inedible, and could serve as a low-cost
and almost ready-to-use substitute for fossil origin diesel. As crude vegetable oil, waste
cooking oil has a high viscosity. Besides, it is enriched with free fatty acids and, hence, can
generate clogging problems in unmodified diesel vehicles, especially in temperate climates
and during the ignition of the engine. Viscosity problems are usually bypassed by blending
WCO with petrol diesel or by using transesterification to produce biodiesel (Pugazhvadivu
et al., 2005; Al-Zuhair et al., 2009; Chen et al., 2009). Al-Zuhair et al. studied the production
of biodiesel with lipases from Candida antarctica and Burkholderia cepacia, both free and
immobilized in ceramic beads, with or without solvents. They found that clay micro-
environments protected immobilized B. cepacia lipase from methanol damage (Al-Zuhair et
al., 2009). Also, Pugazhvadivu et al. proposed solving the injection and filter-clogging
problems by preheating the waste cooking oil (Pugazhvadivu et al., 2005), by comparing the
performance of a diesel engine when using conventional diesel and waste frying oil,
preheated at different temperatures, as fuel. They found that preheating the waste frying oil
to 135ºC improved the overall yield of the engine. In particular, the brake specific energy
consumption and brake thermal efficiency were improved, and the engine exhaust CO and
smoke density were reduced considerably compared to WFO preheated at 75ºC. They
concluded that WFO could be used as a diesel fuel by preheating it to 135ºC.
2.2 Bioethanol and biodiesel
Bioethanol and biodiesel are frequently claimed as the most realistic alternatives to fossil
fuels. These renewable fuels can be extensively produced, and both the fossil fuel
distribution and engines can be easily adapted to work with blends of ethanol and gasoline,

diesel and biodiesel, or even pure ethanol and pure biodiesel (Da Costa et al., 2010). But, in
order to play a significant role in fossil fuel substitution, these renewable fuel industries

Biofuel's Engineering Process Technology

118
should overcome technical limitations in production process efficiency and feedstock-
related issues (UNCTAD, 2010). Decisions about feedstock election, catalysis technology or
energy gain along the production process are of paramount importance for proper biodiesel
and bioethanol production.
2.2.1 Bioethanol and biodiesel production
Bioethanol is produced from simple sugar-rich raw materials or from starchy feedstock,
from which simple sugars can be easily processed and released, which are fermented to
produce ethanol. Bioethanol production comprises three steps. Firstly, the complex sugars
are hydrolysed to release glucose. Subsequently, the glucose is subjected to a second
fermentation step carried out by yeasts such as Saccharomyces cerevisiae; for example,
yielding ethanol and carbon dioxide. The third step consists of a thermochemical process
and is based on the distillation of the diluted ethanol to obtain highly concentrated ethanol.
When using lignocellulosic raw materials such as agricultural residues (corn stover, straw,
sugar cane bagasse), forestry waste, wastepaper and other cellulosic residues, a chemical or
enzymatic hydrolysis pretreatment to degrade the lignin is needed. This additional step
reduces the efficiency of the process. Some improvements have been achieved by the
engineering of cellulases from the Trichoderma genus fungi (Fukuda et al., 2006) and the
utilization of microorganisms able to simultaneously express the cellulase and enzymes
needed for the ethanol fermentation pathway, such as piruvate descarboxilases and alcohol
dehydrogenases (Lu et al., 2006; van Zyl et al., 2007; Jegannathan et al., 2009; Rahman et al.,
2009; van Dam et al., 2009). However, these improvements have still not generated an
efficient and economically affordable process.
With regard to biodiesel, it consists of a mixture of fatty acid alkyl esters (FAAE) obtained
by the transesterification of fatty acids and straight chain alcohols (generally ethanol or

methanol), mainly from vegetable oils. When methanol is the alcohol of choice, the term
used to refer to the biodiesel is fatty acid methyl esters (FAME), while the ethanol-derived
biodiesel is known as fatty acid ethyl esters (FAEE). The properties of the biodiesel obtained
from ethanol or methanol are very similar, but methanol is the preferred alcohol in spite of
its toxicity and fossil fuel origin because of its low cost and wide availability (Ranganathan
et al., 2008; Fjerbaek et al., 2009).
The commercially delivered biodiesel is mainly obtained by the chemical transesterification
of the triglycerides contained in sunflower, rapeseed or palm oil. This process can be carried
out by acid and alkaline liquid catalysts (Kawahara & Ono, 1979; Jeromin et al., 1987; Aksoy
et al., 1988; Fukuda et al., 2001), or heterogeneous solid catalysts such as supported metals,
basic oxides or zeolites (Cao et al., 2008). The preferred catalysts are the liquid ones,
particularly the alkaline ones, because these catalysts are cheap, very versatile and yield less
corrosive fuel than the acid catalysts. Also, liquid catalysts are preferred because the
reusable solid catalysts are still withdrawn with mass transfer and reactant diffusion
problems. However, the alkaline catalysis has several limitations, especially the futile
consumption of the catalyst, problems of viscosity, mass transfer and recovery of biodiesel
and by-products owing to the saponification of the catalyst and free fatty acids in the
presence of water (Freedman et al., 1984; Zhang et al., 2003; Jaruwat et al., 2010). These
problems are bypassed by high temperature reaction conditions, addition of organic
solvents to manage the water presence or enhance the interface formation, or increase of the
alcohol:catalysts ratio (Kawahara & Ono, 1979; Fukuda et al., 2001). Thus, the process
requires high energy inputs to maintain high temperatures conducive to viable

Bioresources for Third-Generation Biofuels

119
transesterification rates, and to separate methanol. Besides, the process generates alkaline
waste water that requires treatment prior to its disposal (Jaruwat et al., 2010). Jointly, all
these negative factors raise doubts about the sustainability and environmental benefits of
the biodiesel industry.

2.2.2 Bioethanol and biodiesel advantages and drawbacks
Extensive bioethanol and biodiesel implantation has been followed by a panoply of
economic, sociopolitical and environmental issues (Guerrero-Compeán, 2008). It is worth
noting the strong dependency of these biofuels industries on crops used for human
nourishment and the feeding of livestock (UNCTAD, 2010). Although a large number of
patents have been proposed to solve many technical problems, the sudden peak in demand
for biofuels has uncovered serious technical limitations of the currently used production
systems. As a consequence, a growing controversy about the real sustainability and
environmental friendliness of the actual biofuels industry has been generated (Fortman et
al., 2008; Abdullah et al., 2009; Demirbaş, 2009; Yee et al., 2009; Jaruwat et al., 2010).
In addition, the consequences of biofuel production for farming practices or food markets,
as well as real greenhouse gases (GHG) emission reduction along the biofuel life cycle,
represent an important issue that, frequently, is not clearly treated. Parameters such as the
kind of biofuel under study, feedstock, and energy inputs needed to maintain the process of
transformation need to be taken into account. Also, the possibility of cogeneration of
electricity or the exchange of energy between the biofuel synthesis and the feedstock
transformation processes must be added to the model. Thus, wide variations in the net
energy gain and consumption of resources can occur owing to the different assumptions
made to calculate the overall benefits and drawbacks. Timilsina and collaborators draw a
general picture of this issue over the OECD estimations. According to these authors, the
most efficient biofuel production scheme is represented by sugarcane-based bioethanol in
Brazil, with a 90% GHG reduction with respect to the gasoline equivalent. This high
efficiency relies mainly on the high yield of this crop and the usage of sugarcane as an
energy source for production plants and the cogeneration of electricity. Second-generation
biofuels based on cellulosic feedstocks present a 70–90% GHG reduction relative to gasoline
or diesel. Combined with electricity cogeneration, this kind of biofuel could have an even
greater effect on GHG reduction, but they are still under development. Ethanol from sugar
beet GHG reduction ranges from 40 to 60%, while wheat-based ethanol presents a 30–50%
GHG reduction. The corn-based production of bioethanol is the least GHG-reducing biofuel
and presents a low efficiency at GHG reductions varying from 0 (even negative in some

cases) to 50% compared to gasoline (OECD,
2008; Timilsina & Shrestha, 2010).
3. Second-generation biofuels
Theoretically, biofuel implantation in transport and industry should solve, or at least
improve, the ecological and economic problems derived from the unsustainability of the
fossil fuel-based energy model.
However, recent field experiences indicate a much more complex scenario. The market
economy and unbalanced relations between different sectors of the economy and national
markets generate unpredictable dynamics of fuels’ raw material prices. In this context, the
development of subsequent new commercial and industrial opportunities has altered the
already unstable behaviour of the agricultural international markets. The sudden peak in
demand for grain, owing to its usage as a raw material for the production of ethanol, has

Biofuel's Engineering Process Technology

120
abruptly increased the prices of corn (Fischer et al., 2009). The demand pressure has
operated similarly in the palm oil market, generating a palm oil tree and soy culture surface
expansion in several regions, with spectacular dimensions in South-East Asia (Abdullah et
al., 2009; Jaruwat et al., 2010), where the biofuels fever threatens biodiversity and has a deep
social impact because of the proliferation of unregulated, intensive, agricultural practices
and the switching of oil usage for traditional human nutrition, housekeeping and livestock
feed (Fortman et al., 2008; Guerrero-Compeán, 2008; Demirbaş, 2009; UNCTAD, 2010; Yee et
al., 2009).
3.1 Feedstock costs and biofuel competition
Biodiesel usually costs over 0.5 US$/l, compared to 0.35 US$/l for petroleum-based diesel
(Demirbaş et al., 2009). It is reported that the high cost of biodiesel is mainly due to the cost
of virgin vegetable oil (Krawczyk, 1996; Connemann & Fischer, 1998). For example, the
soybean oil price is currently 1.27 $/l while the palm oil price is 1.18 $/l (World-Bank, 2011).
Biodiesel from animal fat is currently the cheapest option (0.4–0.5 US$/l), while the

traditional transesterification of vegetable oil is, at present, around 0.6–0.8 US$/l (Bender,
1999). Zhang et al. (2007) stated that there is no global market for ethanol. Within the
reasons for this, crop types, agricultural practices, land labour costs, production plant sizes,
processing technologies and government policies can be cited. The cost of ethanol
production in a dry mill plant currently totals 0.44 US$/l. Corn represents 66% of operating
costs while energy (electricity and natural gas) to fuel the production plant represents nearly
20% of operating costs. Nevertheless, ethanol from sugar cane, produced mainly in
developing countries with warm climates, is generally much cheaper to produce than
ethanol from grain or sugar beet (Bender, 1999). For this reason, in countries like Brazil and
India, sugar cane-based ethanol is becoming an increasingly cost-effective alternative to
petroleum fuels. On the other hand, ethanol derived from cellulosic feedstock using
enzymatic hydrolysis requires much greater processing than from starch or sugar-based
feedstock, but feedstock costs for grasses and trees are generally lower than for grain and
sugar crops. If targeted reductions in conversion costs are achieved, the total cost of
producing cellulosic ethanol in EOCD countries could fall below that of grain ethanol.
Estimates show that ethanol in the EU becomes competitive when the oil price reaches 70
US$/barrel, while in the USA it becomes competitive at 50–60 US$/barrel. For Brazil and
other efficient sugar producing countries such as Pakistan, Swaziland and Zimbabwe, the
competitive ethanol price is much cheaper, between 25–30 US$/barrel. However, anhidrous
ethanol, blendable with gasoline, is still more expensive, although prices in India have
declined and are approaching the price of gasoline. Although the feedstock costs represent
the majority of biofuels’ cost, the production plant size can reduce the final cost of the fuel.
Thus, the generally larger USA conversion plants produce biofuels, particularly ethanol, at
lower cost than plants in Europe. Production costs are much lower in countries with a warm
climate such as Brazil, with less than half the costs of Europe. But, in spite of the reduced
costs of production, ethanol from Brazil is competitive with gasoline owing to the huge
sugar cane production and the cogeneration of electricity (Demirbaş et al., 2009).
3.2 Brazilian and USA models of implementation for the bioethanol industry
Since the Arab oil embargo of the 1970s, Brazil has made an incomparable effort in the
reduction of its energy dependency by intensifying and extending sugar cane-based

bioethanol production. Although the alternative periods of scarcity and abundance of oil

Bioresources for Third-Generation Biofuels

121
have marked fluctuations in the strength of the Brazilian Alcohol National Programme
(Proalcool), the global trend has been an ascending progression in the total production of
alcohol, as well as in the yield per hectare of sugar cane, and the implantation of this alcohol
as transportation fuel. Today, Brazil is the second largest worldwide ethanol producer. In
this way, Brazil has reduced its energy dependency, and has become the first ethanol
exporter. According to Brazilian Government data, this milestone has been achieved on the
basis of rural employment and welfare improvement. The key aspects of the Proalcool
programme are a combination of technological advances, social planification and projection
of the bioethanol industry. According to the Brazilian Government (Da Costa et al., 2010),
owing to the high productivity of sugar cane, Brazil has expanded ethanol production and
use without a significant increment in the fertile land surface used to cultivate sugar cane, or
a food vs. fuel competition. However, there are several authors who are not so enthusiastic
with the success of the Brazilian model, and point to the sugar cane industry as one of the
reasons for the losses in biodiversity and the expansion of agricultural land over doubtfully
catalogued marginal land, which is more relevant and dangerous than the Brazilian
Government data indicates (Coelho et al., 2008; Gauder et al., 2011).
On the other hand, the American bioethanol industry choice of corn grain as its raw material
has been followed by a dramatic rise in the prices of corn derivatives. Although the USA
production of bioethanol supersedes the Brazilian one, the production:consumption ratio of
the former (1:3) is much smaller than the latter (8:3). Despite its commercial orientation, the
global efficiency of the USA model is low compared with the Brazil system and relies on the
high importation taxes that protect the American industry from foreign ethanol inputs (Da
Costa et al., 2010). Finally, the narrow margin of the USA production:consumption ratio
suggests that the model has reached a production glass ceiling that blockades the medium-
term implantation of biofuels in American society and hampers their exportation

(UNCTAD, 2010; Da Costa et al., 2010).
3.3 Europe and Asia: Chemically catalyzed biodiesel
The European and Asian strategy to improve climate change and fossil fuel depletion
problems is based mainly on the chemically catalyzed biodiesel obtained from vegetable
oils. There is a variety of feedstocks for the production of this biofuel, from inedible oils,
(mainly rapeseed oil in Europe or jatropha oil in Asia), to edible oils (principally sunflower
oil in Europe and palm oil or soybean oil in Asia, although corn, peanut, cotton seed or
canola oil can also be cited) (Ranganathan et al., 2008; Abdullah et al., 2009). As the elected
method for industrial biodiesel production is chemical catalysis, these vegetable oils are
preferred to other heterogeneous lipids sources. These other lipids need pretreatment prior
to their use (Peterson, 1986; Fortman et al., 2008), and include waste frying oils, waste-
activated bleaching earth from the oil refinery industry, and even animal origin lipids such
as beef tallow, lard, yellow grease and poultry grease or fat from fat traps, septic tanks, or
waste water sludges. The need for economically viable vegetable oils for biodiesel
production implies the cultivation of greater areas with oil-producing crops such as
sunflowers or palm oil trees. Thus, the previously mentioned rising corn prices, owing to
the derivation of huge amounts of grain for the industrial production of bioethanol, is
neither an isolated case in developing biofuel industries nor the only aspect of the biofuel
industry issue. Like the bioethanol industry, the European and Asian biodiesel industries
have the energy and chemical problems associated with the current biofuels model. These
limitations can be summarized according to nearly obsolete technology, being strongly

Biofuel's Engineering Process Technology

122
dependent on chemical catalysis, non-renewable materials and promotion of non-
sustainable market and farming practices (Guerrero-Compeán, 2008; Demirbaş, 2009;
UNCTAD, 2010).
3.4 Technical aspects of biodiesel production
The industrial production of biodiesel needs to solve several technical problems in order to

obtain this kind of biofuel in an efficient and sustainable way. The physical factors to
consider can be summarized by pH, temperature, hydric activity, solvents and supports.
Depending on the catalyst used to drive the transesterification reaction, some of the cited
factors have different impacts on the global efficiency and feasibility of the process. A non-
optimal configuration of the system can reduce significantly the biodiesel yield and
compromise the viability of the production plant, especially if the upstream by-products,
excess catalyst or auxiliary devices for solvent recovery hinder an easy, clean and rapid
downstream processing of the biofuel.
3.4.1 pH, temperature and hydric activity
As mentioned above, the chemical catalysis of the transesterification reaction requires high
temperatures to achieve an acceptable reaction rate. In the case of the alkaline catalysis, the
minimal temperature to produce conventional biodiesel is 60ºC, while in the acid catalysis
the temperature ranges from 50 to 80ºC (Robles-Medina et al., 2009). Acid catalysis is slower
than the alkaline one and generates a more corrosive fuel, so alkaline catalysts are preferred
by the industry. It incurs a great energy cost in order to initiate and maintain the reaction
(Kawahara & Ono, 1979; Aksoy et al., 1988; Cao et al., 2008). However, the utilization of
sodium hydroxide as a catalyst has a serious limitation in the form of saponification of free
fatty acids if water is present. This drives the increased consumption of the catalyst and
downstream processing problems, such as the separation of glycerol and unreacted
precursors. The solutions to manage this problem include using only virgin oils, often edible
vegetable oils, instead of oils with high free fatty acids and water content, such as waste
cooking oils or animal origin fats, as well as other residual fats. Higher temperatures, up to
120ºC, and the addition of organic solvents, or additional steps for free fatty acids
esterification with sulphuric acid before performing the alkali-catalyzed transesterification
are quite common as well (Jeromin et al., 1987).
When lipases are used as catalysts, it is possible to get over the saponification problems
owing to their ability to transesterificate alcohols with both triacylglycerols and free fatty
acids. Besides, lipases work as well in the presence of water. In fact, they need a certain
hydric activity to maintain their tridimensional structure, so the presence of water is not a
problem with this kind of catalyst — although excessive hydric activity affects the

transesterification reaction because the substrates are water insoluble (Jaeger & Eggert, 2002;
Shah et al., 2004; Gilham & Lehner, 2005; Fjerbaek et al., 2009). Lipases can operate at low or
relatively low temperatures in the range of 20 to 70ºC, and at even lower temperatures if the
enzyme has been obtained from psycrophilic microorganisms (Dabkowska & Szewczyk,
2009). Depending on the chosen lipase and preparation (free, immobilized or whole cell
catalyst), lower temperatures (below 65ºC) can be applied to avoid the thermal denaturation
of the enzyme, thus saving in production costs (Fukuda et al., 2008). Within the
thermostable lipases, we can cite Burkholderia cepacia lipase (Amano PS lipase, from Amano
Pharmaceutical Co., Japan), that reaches its highest activity at 60ºC (Dabkowska &
Szewczyk, 2009), and the lipases obtained from Thermoanaerobacter thermohydrosulfuricus

Bioresources for Third-Generation Biofuels

123
SOL1 and Caldanaerobacter subterraneous subsp. tengcongensis, which show their activity
maximum at 75ºC and tolerate temperatures as high as 95ºC (Royter et al., 2009).
On the other side of the spectrum, the lipase from Bacillus sphaericus MTCC 7526 presents its
optimal temperature at 15ºC, keeping stable until 30ºC, and the Microbacterium phyllosphaerae
lipase presents the optimal temperature at 20ºC and deactivates when the temperature
exceeds 35ºC, with the pH value fixed at 8 for both psycrophilic enzymes (Joseph et al., 2006;
Srinivas et al., 2009). Therefore, pH plays an important role in the enzymatic production of
biodiesel because it influences both the reaction rate and the thermal stability or solvents’
susceptibility of the lipases. An adequate pH can facilitate the optimization of the operation
temperature and improve the activity of the enzyme. Gutarra and collaborators reported a
high stability of the Penicillium simplicissimum lipase in the pH range 4.0–6.0, that showed
the maximal activity at 50ºC and remained stable and active (although with a lower activity)
even at 70ºC (Gutarra et al., 2009).
3.4.2 Heterogeneous catalysts and immobilized enzymes
An alternative to the chemical transesterification of low quality oils with a relatively high
concentration of water or free fatty acids consists of heterogeneous catalysis using acidic

cation-exchange resins, supported metals (Zabeti et al., 2009), basic oxides or zeolites
(Knezevic et al., 1998; Suppes et al., 2004). Even low cost alternatives such as waste eggshell
have been proposed as well (Wei et al., 2009). These kind of catalysts are considered as an
intermediate and relatively low-cost solution between the traditional homogeneous catalysts
and the lipases. However, the cited heterogeneous catalysts are affected by the slow
diffusion of the triglycerydes through their pores and require a higher alcohol:oil ratio to
accelerate the reaction, in order to increase the production (Zabeti et al., 2009). Nevertheless,
the heterogeneous catalysts can serve to improve the reusability and efficiency of
immobilized enzymes and whole cell catalysts. Immobilization of enzymes on inert materials
such as porous ceramic beads (Iso et al., 2001) or polymeric resins (Dizge et al., 2008; Dizge
et al., 2009) can improve their performance. This improvement is owing to the protection
that the pores’ microenvironment brings to the enzyme, avoiding the inhibition or damage
of the enzyme caused by methanol or solvents. Another attractive approach to the
immobilization is the so-called protein-coated microcrystals technology (PCMC). PCMC is
based on the use of crystalized proteins as a support to the lipases, or in the direct use of
crystalized lipases as solid catalysts (Raita et al., 2010). However, the real increase in
reaction rate and enzyme stability with these immobilization techniques is usually lower
than the theoretically expected. One of the reasons for these lower rate issues responds to
the blockage of the pores of the used material as support because of the precipitation of
glycerol or the insufficient circulation of substrates around the enzyme (Zabeti et al., 2009).
The knowledge generated by the intense research on production and use of supports, resins
and porous metallic alloys can be useful for enzymatic production of biodiesel. With this
comparative approach, the optimization of the immobilized enzyme technology could be a
reality in the short rather than medium term.
3.4.3 Alcohol to oil ratio and solvents
Depending on the kind of catalyst used and the selected operation conditions in the
biodiesel production plant, the alcohol to oil molar ratio will present a wide variation.
Adding excess alcohol is a common practice, and could serve as reference. However, excess
alcohol use implies higher reactant associated costs, especially when the alcohol of choice is

Biofuel's Engineering Process Technology

124
ethanol, which is more expensive than methanol. Thus, a more detailed approach to the
system optimization in terms of minimal alcohol consumption is needed. Besides, a fine
adjustment of the alcohol to oil ratio allows the maximal biodiesel production in the shortest
possible time span and with the lowest energy input (Shieh et al., 2003).
The optimization is a relatively simple task when homogeneous catalysts such as sulphuric
acid or sodium hydroxide are used to perform the conventional transesterification of
vegetable oils with methanol. High yields are achieved with a methanol to oil ratio of 1:1
with an alkaline catalyst (although to improve the yield this proportion rises to 6:1) and a
30:1 ratio when an acid catalyst is used (Zhang et al., 2003).
However, in the case of lipase-catalyzed biodiesel, the situation is more complex and the
molar ratio of alcohol to oil varies depending on the type of lipase, the use of an
immobilized or free enzyme, and the alcohol used. Similar to the chemical catalysts, an
increase of the molar alcohol:oil ratio elevates the efficiency of the reaction, but an excessive
alcohol content inhibits and even damages the enzyme, especially when using methanol and
free enzymes. Although the lipase-based solvent-free systems are under intensive research,
owing to advantages such as the direct saving in solvents and the indirect cost reductions in
downstream processes, the utilization of lipases does not necessary mean abandoning the
use of a certain amount of solvents. The addition of solvents like t-butanol, diesel oil, hexane
or dioxane to the precursors of biodiesel usually allows a better mixing of the reactants.
Thus, solvents relieve the problems associated with the different water solubility of lipids
and alcohols. In addition, solvents provide a more durable interaction between the enzyme
and its substrates, and can favour the circulation of reactants through resins and support
pores in immobilized enzyme systems. This improved circulation confers some protection to
the lipases against inhibition by substrates and damages by excessive alcohols. However,
solvents’ addition has to be carefully studied, since an excess of solvent or an inadequate
amount of solvent can affect the enzyme activity and stability. For example, Shieh et al.
studied the optimal operation conditions to transesterificate soybean oil with methanol by

Rhizomucor miehei lipase immobilized on macroporous weak anionic resin beads. They
found that the best transesterification rate was obtained when the methanol:oil molar
proportion was 3.4:1 at 36.5ºC (Shieh et al., 2003). Raita et al. studied the transesterification
of palm oil with ethanol by Thermomyces lanuginosa lipase-coated microcrystals in the
presence of t-butanol. In this case, the optimal conditions were ethanol to fatty acids 4:1
molar ratio and t-butanol:tryacylclycerides 1:1 molar ratio, at 45ºC (Raita et al., 2010.
However, Tongboriboon et al. worked on the solvent-free transesterification of used palm
oil with Thermomyces lanuginosa and Candida antarctica lipases immobilized in porous
polypropylene powder, reporting that the best yield was achieved at an ethanol to oil ratio
of 3:1, and the yield decreased when the molar ratio was increased to 4:1 at 45ºC
(Tongboriboon et al., 2010). These authors pointed to the inhibition of the enzymes by an
excessive amount of ethanol, although it is worth emphasizing that they worked on a
solvent-free system, so the enzyme was relatively vulnerable to alcohol-driven damage. On
the other hand, Shah et al used 4:1 ethanol to oil molar ratio as standard reaction settings in
their study about the transesterification of jatropha oil with ethanol at 40ºC. The
experimental design consisted of a solvent-free system and three different lipases (free and
immobilized on Celite), namely Chromobacterium viscosum, Candida rugosa and Porcine
pancreas lipases, although they did not try different alcohol to oil molar ratios (Shah et al.,
2004).

Bioresources for Third-Generation Biofuels

125
4. Third-generation biofuels
As a response to the problems associated with the recent worldwide implantation of second-
generation biofuels, some authors propose focusing on the processes involved in the
production of such biofuels. This new approach consists of the utilization of microbial
enzymes to achieve the current chemical pretreatment steps of cellulosic or starchy raw
materials (
Carere et al., 2008). Microorganisms deal with the degradation of lignocellulose,

hemicellulose or lipid-rich materials by means of enzyme catalyzed processes at near to
room temperature. Therefore, microbial enzymes could be used to make the current biofuels
industry cleaner and greener. Furthermore, the production of biofuels would be coupled
with the management of woody and oily wastes, converting these residues into suitable and
cheap raw materials (Steen et al., 2010).
4.1 Microalgae-based biodiesel production
Another promising lipids source, still not implemented but currently being studied
worldwide, is represented by microalgae. Microalgae have a high potential as biodiesel
precursors because many of them are very rich in oils, sometimes with oil contents over 80%
of their dry weight, although not all species are suitable as biodiesel production oils (Chisti,
2008; Manzanera, 2011). Besides, these microorganisms are able to double their biomass in
less than 24 hours, achieving a reduction between 49 and 132 fold in the medium culture
time required by a rapeseed or soybean field. Furthermore, microalgae cultures require low
maintenance and can grow in wastewaters, non-potable water or water unsuitable for
agriculture, as well as in seawater (Mata et al., 2010). The production of microalgae biodiesel
could be combined with the CO
2
removal from power generation facilities (Benemann,
1997), the treatment of waste water from which microalgae would remove NH
4
+
, NO
3
-
and
PO
4
3-
(Aslan & Kapdan, 2006), or the synthesis of several valuable products, from bioethanol
or biohydrogen to organic chemicals and food supplements (Banerjee et al., 2002; Chisti,

2007; Rupprecht, 2009; Harun et al., 2010). However, microalgae biomass-based biofuels
have several problems ranging from the optimization of high density and large surface units
of production to the location of the microalgae production unit. Anyway, the main decisions
to take are the adoption of open or closed systems, and the election of batch or continuous
operation mode. As will be discussed below, depending on the system and mode of
operation choice, there will be different advantages and drawbacks.
4.1.1 Open vs. closed systems
Microalgae can be cultivated in open-culture systems such as lakes or (raceway) ponds, and
in closed-culture systems called photobioreactors (PBRs). Open-culture systems are
normally cheaper to build and operate, more durable and have a higher production capacity
than PBRs. However, open systems are more energy expensive in terms of nutrient
distribution owing to mass transfer problems, and have their depth limited to 15 cm, to
ensure that the microalgae receive enough light to grow. Moreover, ponds are more
sensitive to weather changes, and temperature, evaporation and light intensity controls are
not feasible. Furthermore, these open systems require more land area than PBRs, and are
more susceptible to contamination, both from bacteria and from microalgae present in the
surroundings of culture installations (Manzanera, 2011).
In contrast, PBRs are more flexible and are intensive land-usage systems that can be
configured according to the specific physical-chemical requirements of the algae of choice,

Biofuel's Engineering Process Technology

126
allowing the cultivation of species unsuited to open ponds. Nutrient homogenization, light
distribution, pH, temperature, CO
2
and O
2
control can be achieved in photobioreactors.
Thus, closed systems provide more stable and appropriate growing conditions, allowing

higher cell densities and minimizing contamination. Nevertheless, PBRs have several
technical problems that make them non-competitive in applications that can be achieved in
raceway ponds. Such problems are overheating, bio-fouling, shearing stress, oxygen
accumulation, scaling-up difficulties and the high costs of building, operation and
maintenance (Chen et al., 2011).
Within these problems, it is worth highlighting capital building investment and high operation
costs. PBRs biomass production costs may be one order of magnitude higher than in open
systems. If the biomass added value is high, PBRs can be competitive. Otherwise, open ponds
will be the preferred option. However, the evaluation of performance of open and closed
systems is complex and depends on several factors, such as algal species or productivity
computation method. Three parameters are commonly used to evaluate productivity in
microalgae cultivation installations. Firstly, volumetric productivity (VP), that is, productivity
per unit of reactor volume (g/l· d). The second parameter is area productivity (AP), defined as
productivity per unit of ground area occupied by the reactor (g/m
2
· d). The third one is
illuminated surface productivity (ISP), namely the productivity per unit of reactor illuminated
surface area (g/m
2
· d). Nevertheless, the election of closed or open systems relies on more
aspects apart from productivity, as will be discussed below (Richmond, 2010).
4.1.2 Continuous vs. batch operation mode
PBRs can be operated in batch or continuous mode. There are several advantages when
using them in continuous mode. Firstly, continuous culture provides a higher control than
batch mode. Secondly, growth rates can be regulated and keep in a steady state for long
periods, and the biomass concentration can be modulated by dilution rate control. In
addition, results are more reliable and reproducible owing to the steady state of continuous
reactors, and the system yields better quality production (Molina et al., 2001).
However, there are limitations that can make the continuous process unsuitable for some
cases. One of these limitations is the difficulty in controlling the production of some non-

growth-related products. For instance, the system often requires feed-batch culturing and
continuous nutrient supply that can lead to wash-out. Filamentous organisms can be
difficult to grow in continuous PBRs because of the viscosity and heterogeneity of the
culture medium. Another problem is that the original strain can be lost if it is displaced by a
faster-growing contaminant. The contamination risk and loss of reliability of the bioreactor
becomes more relevant when long incubation periods are needed, so the potential initial
investment in necessary better quality equipment could rise and hamper the economic
viability of the production unit (Mata et al., 2010).
The possible coproduction of high value chemicals could lead to the solution of the above
problems, but it implies taking multiple parameters and options into consideration. The
microalgae production units will suffer drastic changes, both in the operational aspect
(temperature, insolation, wind, microalgal and bacterial or fungical contaminations etc.) and
in the commercial one (oscillations in value of by-products, improvements in centrifugation
or extraction strategies or development of non-algal biofuels, etc). Taking into consideration
all the above mentioned parameters, it can be ascertained that any microalgae-based
biodiesel project is unique. Hence, such projects must be designed by thinking in terms of a
flexible or even multipurpose and adaptable installation (Richmond, 2010).

Bioresources for Third-Generation Biofuels

127
4.2 Biodiesel production from oily biomass
Microalgae are not the only option to produce biofuels from oily biomass. Multiple
prokaryotes and eukaryotes can accumulate high amounts of lipids. But, as occurred with
microalgae, not all species are suitable for biodiesel production owing to differences in the
kind of storage lipids. Thus, as stated by Waltermann & Steinbüchel (2010), many
prokaryotes synthesize polymeric compounds such as poly(3-hydroxybutyrate) (PHB) or
other polyhydroxyalkanoates (PHAs), whereas only a few genera show accumulation of
triacylglycerols (TAGs) and wax esters (WEs) in the form of intracellular lipid bodies. On
the other hand, storage TAGs are often found in eukaryotes, while PHAs are absent, and

WE accumulation has only been reported in jojoba (Simmondsia chinensis). All these lipids
are energy and carbon storage compounds that ensure the metabolism viability during
starvation periods. Similar to the formation of PHAs, TAGs and WE, synthesis is promoted
by cellular stress and during imbalanced growth; for instance, by nitrogen scarcity alongside
the abundance of a carbon source (Kalscheuer et al., 2004).
The most interesting prokaryote genera in terms of accumulation of TAGs are
nocardioforms such as Mycobacterium sp., Nocardia sp., Rhodococcus sp., Micromonospora sp.,
Dietzia sp., and Gordonia sp, alongside streptomycetes, which accumulate TAGs in the cells
and the mycelia. TAGs storage is also frequently shown by members of the gram-negative
genus Acinetobacter (although, in this case, WE are the dominant inclusion bodies
components) (Waltermann & Steinbüchel, 2010). Within eukaryotes, with the exception of
algae, yeasts of the genera Candida (non albicans) (Amaretti et al., 2010), Saccharomyces
(Kalscheuer et al., 2004; Waltermann & Steinbüchel, 2010) and Rhodotorula (Cheirsilp et al.,
2011) are the most interesting ones to produce biodiesel feedstocks.
Steinbüchel and collaborators have worked on the heterologous expression of the non
specific acyl transferase WS/DGAT from Acinetobacter calcoaceticus ADP1 in Saccharomyces
cerevisiae H1246 (a mutant strain unable of accumulating TAGs) (Kalscheuer et al., 2004).
These authors found that the yeast recovered the ability to accumulate TAGs, as well as fatty
acid ethyl esters and fatty isoamyl esters. This finding showed that the Acinetobacter
calcoaceticus transferase had a high potential for biotechnological production of a large
variety of lipids, either in prokaryotic and eukaryotic hosts. From this basis, as will be
discussed in detail in Section 4.3, they worked on Escherichia coli TOP 10 (Invitrogen) and
obtained an engineered strain able to produce fatty acid ethyl esters (biodiesel) directly from
oleic acid and glucose (Kalscheuer et al., 2006).
Another possibility is combining the biomass obtained from microalgae and yeast, as
recently proposed by Cheirsilp et al. (2011). These authors studied a mixed culture of
oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris in industrial wastes.
The used effluents, including both a seafood processing wastewater and molasses from a
sugar cane plant. They found a synergistic effect in the mixed culture. R. glutinis grew faster
and accumulated more lipids in the presence of C. vulgaris, that acted as an oxygen

generator for yeast, while the microalgae obtained surplus CO
2
from yeast. The optimal
conditions for lipid production were 1:1 microalga to yeast ratio initial pH of 5.0, molasses
concentration at 1%, 200 rpm shaking, and light intensity at 5.0 klux under 16:8 hours light
and dark cycles (Cheirsilp et al., 2011).
4.3 Whole cell catalysts
Pure or immobilized enzymes obtained from microorganisms could reduce the energy costs
of industrial ethanol and biodiesel production. Nevertheless, the cellulases used to treat

Biofuel's Engineering Process Technology

128
(ligno)cellulosic materials such as forestry residues, waste paper or straw are difficult to
purify, like the lipases used for the transesterification of lipids yielding biodiesel. Hence,
their price is still too high to make their usage economically viable (Shieh et al., 2003;
Ranganathan et al., 2008). Another limiting factor for the use of enzymes is the inactivation
and inhibition by reactants and substrates. These drawbacks are the object of an intensive
effort to make possible the reutilization of enzymes through protein engineering
(Ebrahimpour et al., 2008), in order to increase their stability and activity. Research interest
is also targeted on immobilization in different supports or the usage of genetically
engineered microorganisms, called whole cell catalysts, which carry the necessary enzymes,
avoiding their exposure to inhibiting substrates and operating as microrefineries
(Kalscheuer et al., 2006). In the case of biodiesel microbiological production that will be
revealed in detail below, the authors proposing and developing this technology refer to this
third-generation biofuel as ‘Microdiesel’. The microbial production of biodiesel requires the
construction of genetically modified microorganisms, able to transesterificate ethanol with
lipids and, if possible, able to produce it by themselves to optimize the whole process. Since
their 2006 work on microdiesel production on the laboratory scale using an engineered
Escherichia coli strain, Steinbüchel and collaborators have established the guidelines of

microdiesel industry development. Their approach consisted of expressing heterologously
in E. coli the genes from Zymomonas mobilis, encoding for piruvate decarboxylase (pdc) and
alcohol dehydrogenase (adhB), as well as the Acinetobacter baylyi non specific acyl transferase
ADP1 (atfA). The obtained strain was able to carry out the aerobic ethanol fermentation from
sugars, as well as the enzymatic transesterification of this alcohol with the fatty acids
derived from the lipidic metabolism, yielding FAEE, referred to as ‘microdiesel’ by the
authors (Kalscheuer et al., 2006). Recently, Elbahloul and Steinbüchel have used the
aforementioned microdiesel producing E. coli at a pilot plant scale, using glycerol and
sodium oleate as carbon and fatty acids sources respectively, with promising results
(Elbahloul & Steinbüchel, 2010). Nevertheless, their conclusions for both studies indicate
that there is still a long way to go to the industrial application of their findings, and that the
technique needs to be modified to make the engineered strains adaptable to different lipids
rich sources and to lignocellulosic raw materials. These modifications would allow the
usage of forestry and agricultural wastes, making the biodiesel production process at least
as versatile as chemical transesterification.
4.4 Microdiesel production from residues
Vegetable oils are expensive and require large areas of farmland for their production, so the
direct usage of these oils for biodiesel production is expensive and unsustainable. However,
there are multiple and as yet unexploited alternative fatty acid sources. Similarly, bioethanol
production for its direct use as a biofuel or as a biodiesel precursor requires huge amounts
of corn grain or sugar cane. Nevertheless, industrial residues such as the vegetable oil
refinery waste, as well as farming, forestry, livestock and domestic solid and liquid waste
(Chen et al., 2009; Dizge et al., 2009) are widespread and huge sources of lipids and carbon.
Wang et al. proposed the soybean oil deodorizer distillate (SODD), a by-product from the
soybean oil refineries that represents 0.3–0.5% of the soybean oil processed, to produce
biodiesel. With 45–55% of triglycerides and 25–35% of free fatty acids, these authors
estimated that around 80% of the SODD can be transformed into biodiesel in a
transesterification with methanol by the Thermomyces lanuginosa and Candida antarctica
lipases in the presence of tertbutanol and 3Å molecular sieve (Wang et al., 2006). Park et al.

Bioresources for Third-Generation Biofuels

129
used waste-activated bleaching earth (ABE), a residue of the rapeseed or palm oil refinery
industry that stores 35–40% of oil and can be used to synthesize multiple bulk chemicals,
including biodiesel. As in the Wang example, these authors chose methanol as alcohol, but
their solvent choice was fuel oil and kerosene, the catalyst was Candida cylindracea lipase and
the obtained FAME was extracted with a filter press (Park et al., 2008). Al-Zuhair and
colleagues studied the production of biodiesel from simulated waste cooking oil (SWCO)
with free- and immobilized- on ceramic beads Candida antarctica and Burkholderia cepacia
lipases, with or without organic solvent. They obtained the best yield when they used B.
cepacia without organic solvent, and observed that the system worked better when the
enzymes were immobilized, probably because the clay structural microenvironments
offered the lipases protection against the methanol derived denaturation (Al-Zuhair et al.,
2009). Recently, Steen et al., among others, have proposed the direct fermentation of
cellulosic biomass to produce biodiesel, fatty alcohols, waxes and other valuable chemicals
(Steen et al., 2010). Their approach combines the waste management and the guidelines
defined by Steinbüchel et al. with the new trends in synthetic biology and consolidated
bioprocesses. This multidisciplinary approach brings a new flexible, easy-to-modify toolbox,
composed of genetically modified FAEE synthetic strains, harbouring the enzymatic
apparatus needed to produce ethanol from raw (hemi)cellulosic materials, to
transesterificate it with fatty acids, or to synthesize both the fatty acids and the ethanol
directly from the cellulose (Steen et al., 2010).
4.5 Wastewater sludges-based microdiesel
The microdiesel concept initiated by Steinbüchel et al. can be combined with the
management and reutilization of waste waters by the application of microbial lipases to
transesterificate the lipids present in the dairy industry or urban wastewater sludges. The
lipidic fraction of sludges from urban wastewater treatment represents between 17 and 30%
of the dry weight. This lipidic fraction is formed by direct absorption of fats present in the
water by the sludge particles and by the phospholipids released from the cell membranes of

micro-organisms, as well as from metabolites and cell lysis by-products (Boocock et al., 1992;
Shen & Zhang, 2003; Jardé et al., 2005).
Lipid-rich wastewaters require pretreatment in order to reduce the amount of lipids and
ease the subsequent conventional treatment. The pretreatment is usually based on physical
processes, the most common of which are fat traps, tilted plate separators (TPS), and
dissolved air flotation (DAF) units. In addition, centrifuges and electroflotation systems are
used occasionally (Willey, 2001). Fat traps are rectangular or circular vessels through which
the wastewater passes under laminar-flow conditions, at a rate that allows the lipids to rise
to the surface near to the outlet end of the trap. The separation principle is based on Stoke's
law, relating rising velocity of a particle to its diameter, so the theoretical separation
efficiency is dependent on depth. In practice, fat traps have a depth of 1.5 m, although if the
accumulation of a bottom sludge is expected, then an additional 0.5 m would be added to
the total liquid depth. Gravity flow is preferred to pumping when feeding the trap, in order
to minimize the wastewater emulsification. Fat traps are used in the food industry and in
restaurants (Willey, 2001).
Meanwhile, tilted plate separators were developed in the petrochemical industry and are
based on the fact that surface area, rather than depth, determines the oil separation. The
introduction of tilted plates into a vessel provides many parallel gravity separators with a

Biofuel's Engineering Process Technology

130
high surface to volume ratio in a shallow tank. Typically, TPS can occupy less than 10% of
the area needed to install a conventional fat trap, although they have some disadvantages.
They are susceptible to fouling if solid or semi-solid fat is present in the effluent and a crane
is required to remove the plate pack for cleaning. Besides, the pumping systems have to be
carefully selected and controlled to avoid surging and liquid depth fluctuations (Zeevalkink
& Brunsmann, 1983; Willey, 2001). Finally, dissolved air flotation units are based on the
flotation of lipids by means of microbubble clouds (60-70 m bubble diameter) created by
the injection into water of 6 bar pressure air through nozzles. Microbubbles attach to the

surface of the fat and oil particles, increasing their rise rate. These systems are used both in
the food industry (Willey, 2001) and in the mining wastewater treatment (Tessele, 1998).
Once the pre-treatment has finished, wastewater can receive further treatment prior to its
disposal or biological treatment. Thus, chemical treatment may be used to reduce the total
fatty matter in wastewater. Such treatment uses aluminium sulphate, ferric chloride, or
more usually, lime, to break the emulsion and coagulate the fat particles. Subsequently, the
fats can be separated by flotation or sedimentation. The rate of sedimentation can be
improved by a second-stage flocculation, involving the addition of low levels of
polyelectrolyte (0.5-5.0 mg/l) to the wastewater once coagulation has taken place (Willey,
2001).
The use of sludges to produce biofuels is not a new idea itself, but the available literature
focuses mainly on the methane production by anaerobic fermentation, currently applied in
the majority of waste water treatment plants (WWTP) to provide energy to these
installations, or for fermentative biohydrogen production, which is still not industrially
available (van Groenestijn et al., 2002; Wang et al., 2003). Several groups have studied the in
situ transesterification of WWTP sludges, but have focused on the chemical catalysis of the
transesterification with methanol (Haas & Foglia, 2003; Mondala et al., 2009). However this
method still presents the same limiting factors that affect the chemical transesterification of
edible vegetable oils (Freedman et al., 1984). In spite of their chemical approach, these works
provide useful information about several aspects of the biodiesel production process,
especially at the first stages of the process. Thus, one common problem of chemical and
enzymatic biodiesel production is the need for the pretreatment of the feedstock to make its
lipids easily available to the catalyst. In the case of wastewater treatment sludge, this
pretreatment step usually implies the use of organic or non-polar solvents to release the
lipids from the organic matter (Antczak et al., 2009; Siddiquee & Rohani, 2011).
The most extended protocols rely on chloroform:methanol mixtures, as used in the Folch's
method (Folch, 1957), in which a 2:1 chloroform:methanol reactant is mixed with the sample,
where water acts as ternary component to form an emulsion. After equilibration with a
fourth volume in saline solution, the emulsion separates in two phases: the lower one
containing chloroform:methanol:water in the proportions 86:14:1 alongside the lipids; and

the upper one containing the same solvents in proportions 3:48:47 and carrying the non-
lipidic components of the sample. Bligh and Dyer's method is a simplified variant of the
former, but requires the re-extraction of the sample residue with chloroform (Bligh & Dyer,
1959). Nevertheless, there are some methods with near to Folch's reagent yielding which use
less toxic reagents, such as pure hexane or different combinations of hexane and other
solvents, such as the hexane-isopropanol (3:2) blend proposed by Hara and Radin (Hara &
Radin, 1978), or the ethyl acetate-ethanol (2:1) mixture used by Lin et al. (Lin et al., 2004). For

Bioresources for Third-Generation Biofuels

131
a detailed revision of the solvents based extraction protocols, see Kuksis, 1994; Murphy,
1994; Kates, 1996.
In spite of being slightly less toxic than chloroform, the cited solvents are hazardous and
present enough management risks to consider other extraction strategies. Several authors
propose solvent-free methods based on ionic liquids (Ha et al., 2007), boiling the sludge or
subjecting it to supercritical gases, mainly t-butanol (Wang et al., 2006; Royon et al., 2007),
propane (Rosa et al., 2008), syngas (Tirado-Acevedo et al., 2010) and CO
2
(Helwani et al.,
2009), or even to extreme pressures and temperatures (cracking) (Saka & Kusdiana, 2001).
All of them are costly and not feasible with the current technology (Siddiquee et al., 2011). A
more realistic and ready-to-use option is extraction using hot ethanol, which can be used to
perform the lipids’ extraction without using coadyuvant solvents. This approach to
extraction can be illustrated with the works developed by Holser and Akin (2008) or Nielsen
and Shukla (2004), among others. Although these authors have focused on the ethanol-
based extraction of high value lipids from flax processing wastewater and egg yolk powder,
respectively, their findings could be scaled and applied to biodiesel production from
wastewater sludges. Nielsen and Shukla found that the use of ethanol at room temperature
led to the extraction of nearly all the phospholipids, together with cholesterol and a minor

part of the triacylglycerols, without special extraction and filtration devices. On the other
hand, Holser and Akin performed a serial extraction of the lipids present in flax wastewater
in three steps, under different temperature values (50, 80, 90 and 100ºC). They found that the
most efficient extraction was achieved when the sample-ethanol mixture was heated to 90ºC
and the reaction time was 15 minutes (Holser & Akin, 2008).
Considering the above findings, and taking into account the fact that the enzymatic
production of biodiesel generally requires high alcohol to oil ratios, to improve the
solubilization of the lipids and the formation of water-oil, enzyme-oil and enzyme-alcohol-
oil interfaces, we propose that a suitable scheme for the production of biodiesel from WWTP
sludge could be as simple as using a pressurized pretreatment tank, where the sludge is
soaked in ethanol, kept at 90ºC under stirring and refluxed to subject the mixture to three
extraction cycles. This is followed by incubation in a reaction tank where the extracted
lipids, alongside with part of the ethanol used in the previous step, are added to a reaction
mix containing the enzyme (free, immobilized or whole cell catalyst) and kept at the optimal
temperature and pH conditions, to ensure both enzyme stability and an acceptable
microdiesel production rate. Heat exchangers between the two tanks could serve to save
energy, using the heat released before entering the second tank to preheat the sludge before
entering the first one.
The system could even be autonomous in terms of ethanol requirements if the engineered
microorganism used to produce the lipase was able to produce ethanol simultaneously, or if
the cited tanks were coupled with a third reactor where bioethanol was produced from
sugars present in the non-lipidic products obtained in the pretreatment tank by means of
ethanol-producing yeast or bacteria strains. In the case of economic restrictions, some short-
term cost reduction could be achieved by replacing the pretreatment pressurized tank with a
non-pressurized unit, and keeping the temperature of the extraction mix below 79ºC,
although it would imply medium-term economic losses because the lower extraction
efficiency must be compensated by performing more extraction cycles at a higher reflux rate
and a greater ethanol volume in the pretreatment tank, or even by the use of at least two
serial pretreatment tanks.

Biofuel's Engineering Process Technology

132
5. Conclusion
As a short-term response to the consequences of greenhouse gas emissions and the
unsustainability of the fossil fuel-based energy model, the industry has developed ready-to-
use substitutes for traditional fossil fuels, delivered generally and ambiguously under the
commercial ‘bio’ denomination. However, the first- and second-generation of so-called
biofuels are neither of completely biological origin nor based on renewable and
environmentally friendly feedstocks. In addition, the production techniques rely on high
energy inputs, both in feedstock production (as is the case for rapeseed, soybean or palm oil)
and in the biofuel synthesis (acid catalyzed biodiesel or corn bioethanol perfectly illustrate
the neat energy gain problems). Alongside these problems, new and complex problems have
emerged. Firstly, the increase in the prices of grain and vegetable oils used both to produce
biofuels and for human nourishment and livestock feeding; and secondly, the expansion of
agricultural land to increase production of sugar cane or vegetable oils to satisfy the huge
demand for these sugar and lipid sources, generated by the abrupt increase in biofuels
production. Thus, the development of cleaner and more sustainable biofuels is required to
achieve the challenge to totally replace traditional fossil fuels by third-generation biofuels,
independent of non-renewable precursors or inefficient industrial processes, that damage
the environment directly and indirectly and threaten biodiversity and food security
(UNCTAD, 2010).
A great variety of domestic, agricultural and industrial residues, from lignocellulosic
forestry and agriculture waste to fatty acid rich waste waters, generated by the dairy,
poultry or vegetable oil refinery industries, as well as the sludges from urban waste waters,
can be used as precursors of biofuels. The treatment of these residues could be combined
with the production of third-generation biofuels by enzymatic catalysis because the high
cost of enzymes could be compensated by the low cost of the residues (or even the presence
of incentives for residue reduction and management). But the massive application of these
concepts requires a series of technical and biotechnological improvements, such as the

optimization of lipids and sugars extraction, feedstock pretreatment processes, biofuels
production plant design, heterogeneous catalysts and enzyme immobilization techniques,
protein engineering of lipases, alcohol dehydrogenases or hydrolases to increase their
activity and reusability, genetic engineering of microbes to facilitate both the pretreatment
of precursors, and the synthesis and purification of the biofuels.
6. Acknowledgement
We thank the Junta de Andalucía (Spain) for funding this study through project reference
P08-RNM-04180 and the Spanish Ministry of Science and Technology for funding through
project reference CTM2009-09270. M. Manzanera received grants from the Programa Ramon
y Cajal, (Ministerio de Educacion y Ciencia MEC, Spain, and ERDF, European Union).
7. References
Abdullah, A.Z., Salamatinia, B., Mootabadi, H. & Bathia, S. (2009). Current status and
policies on biodiesel industry in Malaysia as the world's leading producer of palm
oil. Energy Policy 37(12): 5440-5448

Bioresources for Third-Generation Biofuels

133
Aksoy, H.A., Kahraman, I., Karaosmanoglu, F., & Civelekoglu, H. (1988). Evaluation of
Turkish sulphur olive oil as an alternative diesel fuel. Journal of the American Oil
Chemists Society 65: 936-938.
Alauddin, Z.A.B.Z., Lahijani, P., Mohammadi, M., & Mohamed, A.R. (2010). Gasification of
lignocellulosic biomass in fluidized beds for renewable energy development: A
review. Renewable and Sustainable Energy Reviews 14: 2852-2862.
Al-Zuhair, S., Dowaidar, A. & Kamal, H. (2009). Dynamic modelling of biodiesel production
from simulated waste cooking oil using immobilized lipase. Biochemical Engineering
Journa.l 44: 256-262.
Amaretti, A., Raimondi, S. & Rossi, M. (2010). Production of Single Cell Oils from Glycerol
By Oleaginous Yeasts. Journal of Biotechnology 150: 389.
Antczak, M.S., Kubiak, A., Antczak, T., & Bielecki, S. (2009). Enzymatic biodiesel synthesis:

Key factors affecting efficiency of the process. Renewable Energy. 34: 1185-1194.
Aslan, S., & Kapdan, I.K. (2006) Batch kinetics of nitrogen and phosphorus removal from
synthetic waste water by algae. Ecological Engineering. 28: 64-70.
Banerjee, A., Sharma, R., Chisti, Y., & Banerjee, U.C. (2002). Botryococcus braunii: A
renewable source of hydrocarbons and other chemicals. Critical reviews in
Biotechnology. 22: 245-279.
Bender, M. (1999). Economic feasibility review for community-scale farmer cooperatives for
biodiesel. Bioresource Technology. 70: 81.
Benemann, J.R. (1997). CO
2
mitigation with microalgae systems. Energy Conversion and
Management. 38: S475-S479.
Bligh, E.G. & Dyer, W.J. (1959). A rapid method of total lipid extraction and purification.
Canadian Journal of Biochemistry and Physiology. 37: 911-917, 1959.
Bludowsky, T. & Agar, D.W. (2009). Thermally integrated bio-syngas-production for
biorefineries. Chemical Engineering Research and Design. 87: 1328.
Boocock, D.G.B., Konar, S.K., Leung, A. & Ly, L.D. (1992). Fuels and chemicals from sewage
sludge. The solvent extraction and composition of a lipid from a raw sewage
sludge. Fuel. 71: 1283.
Cao, F., Chen, Y., Zhai, F., Li, J., Wang, J., Wang, X., Wang, S., & Zhu, W. (2008). Biodiesel
production from high acid value waste frying oil catalyzed by superacid
heteropolyacid. Biotechnology and Bioengineering. 101: 93- 100.
Carere, C.R., Sparling, R., Cicek, N. & Levin, D.B. (2008). Third-generation biofuels via direct
cellulose fermentation. International Journal of Molecular Sciences. 9: 1342-1360.
Cescut, J., Severac, E., Molina-Jouve, C. & Uribelarrea, J.L. (2011). Optimizing pressurized
liquid extraction of microbial lipids using the response surface method. Journal of
Chromatography A. 1218: 373.
Cheirsilp, B., Suwannarat, W. & Niyomdecha, R. (2011). Mixed culture of oleaginous yeast
Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from
industrial wastes and its use as biodiesel feedstock. New Biotechnology In Press,

Corrected Proof.
Chen, Y., Xiao, B., Chang, J., Fu, Y., Ly, P. & Wang, X. (2009). Synthesis of biodiesel from
waste cooking oil using immobilized lipase in fixed bed reactor. Energy Conversion
and Management. 50: 668-673.

Biofuel's Engineering Process Technology

134
Chen, C-Y., Yeh, K-L., Aisyah, R., Lee, D-J. & Chang, J-S. (2011). Cultivation, photobioreactor
design and harvesting of microalgae for biodiesel production: A critical review.
Bioresource Technology. 102: 71.
Chisti, Y. (2008). Biodiesel from microalgae beats bioethanol. Trends in Biotechnology. 26: 126-
131.
Chisti, Y. (2007). Biodiesel from microalgae. Biotechnology Advance., 25: 294-306.
Chisti, Y. (1992). Assure bioreactor sterility. Chemical Engineering Progress. 88: 80-85.
Choi, K.H., Chisti, Y. & Moo-Young, M. (1995). Split-channel rectangular airlift reactors:
Enhancement of performance by geometric modifications. Chemical Engineering
Communications. 138: 171-181.
Coelho, C.H., Francisco, J.G., Nogueira, R.F.P. & Campos, M.L.A.M. (2008). Dissolved
organic carbon in rainwater from areas heavily impacted by sugar cane burning.
Atmospheric Environment, 42: 7115.
Connemann, J., & Fischer, J. (1998). Biodiesel in Europe 1998: Biodiesel Processing
Technologies. Paper presented at the International liquid biofuels congress. July 19-22,
1998, Curitiba-Parana, Brazil
Conti, J.J. (2009). Annual Energy Outlook 2009 With Projections to 2030. USDE, EIA. Energy
Information Administration Office of Integrated Analysis and Forecasting US.
Department of Energy Washington, DC 20585.
Da Costa, A.C.A., Junior, N.P. & Aranda, D.A.G. (2010). The situation of biofuels in Brazil:
New generation technologies, Renewable and Sustainable Energy Reviews. 14: 3041-
3049.

Dabkowska, K. & Szewczyk, K.W. (2009). Influence of temperature on the activity and
enantioselectivity of Burkholderia cepacia lipase in the kinetic resolution of mandelic
acid enantiomers. Biochemical Engineering Journal 46: 147-153.
Demirbaş, A. (2009). Political, economic and environmental impacts of biofuels: A review.
Applied Energy, 86: S108-S117.
Dizge, N., Aydiner C., Imer D.Y., Bayramoglu, M., Tanriseven, A. & Keskinler, B. (2009).
Biodiesel production from sunflower, soybean, and waste cooking oils by
transesterification using immobilized lipase. Bioresource Technology, 100: 1983-1991.
Dizge, N. & Keskinler, B. (2008). Enzymatic production of biodiesel from canola oil using
immobilized lipase. Biomass and Bioenergy, 32: 1274-1278.
Domínguez, A., Fernández, Y., Fidalgo, B., Pis, J.J. & Menéndez, J.A. (2008). Bio-syngas
production with low concentrations of CO
2
and CH
4
from microwave-induced
pyrolysis of wet and dried sewage sludge. Chemosphere, 70: 397.
Ebrahimpour, A., Rahman, R.N.Z.R.A., Ch'ng, D.H.E., Basri, M. & Salleh, A.B. (2008). A
modelling study by response surface methodology and artificial neural network on
culture parameters optimization for thermostable lipase production from a newly
isolated thermophilic Geobacillus sp. strain ARM. BMC Biotechnology. 8: 96.
Elbahloul, Y. & Steinbüchel, A. (2010). Pilot-scale production of fatty acid ethyl esters by an
engineered Escherichia coli strain harboring the p(Microdiesel) plasmid. Applied and
environmental microbiology. 76: 4560-4565.
Fischer, G., Hizsnyik, E., Prieler, S., Shah, M. & van Velthuizen, H. (2009). Biofuels and food
security. Prepared by the International Institute for Applied Systems Analysis
(IIASA) for OPEC Fund for International Development (OFID). OFID Pamphlet
Series.

Bioresources for Third-Generation Biofuels

135
Fjerbaek, L., Christensen, K.V. & Norddahl, B. (2009). A review of the current state of
biodiesel production using enzymatic transesterification. Biotechnology and
Bioengineering 102: 1298-1315.
Folch, J., Lees, M. & Sloane-Stanley, G.H. (1957). A simple method for the isolation and
purification of total lipides from animal tissues. The Journal of Biological Chemistry,
226: 497-509.
Fortman, J.L., Chhabra, S., Mukhopadhyay, A., Chou, H., Lee, T.S., Steen, E. & Keasling, J.D.
(2008). Biofuel alternatives to ethanol: Pumping the microbial well. Trends in
Biotechnology. 26: 375-381.
Freedman, B., Pryde, E.H. & Mounts, T.L. (1984). Variables affecting the yields of fatty esters
from transesterified vegetable oils. Journal of the American Oil Chemists Society. 61:
1638-1643.
Fukuda, H., Kondo, A. & Noda, H. (2001). Biodiesel fuel production by transesterification of
oils. Journal of Bioscience and Bioengineering. 92: 405-416.
Fukuda, H., Hama, S., Tamalampudi, S. & Noda, H. (2008). Whole-cell biocatalysts for
biodiesel fuel production. Trends in Biotechnology. 26: 668-673.
Fukuda, T., Ishikawa, T., Ogawa, M., Shiraga, S., Kato, M., Suye, S-I. & Ueda, M. (2006)
Enhancement of cellulase activity by clones selected from the combinatorial library
of the cellulose-binding domain by cell surface engineering. Biotechnology progress
22: 933-938.
Gauder, M., Graeff-Hönninger, S. & Claupein, W. (2011). The impact of a growing
bioethanol industry on food production in Brazil. Applied Energy. 88: 672.
Gilham, D. & Lehner, R. (2005). Techniques to measure lipase and esterase activity in vitro.
Methods. 36: 139-147.
Grothe, E. & Chisti, Y. (2000). Poly(-hydroxybutyric acid) thermoplastic production by
Alcaligenes latus: Behavior of fed-batch cultures. Bioprocess Engineering. 22: 441-449.
Guerrero-Compeán, R. (2008). Regional, economic, and environmental effects of traditional
and biotechnologically enhanced ethanol production processes in Brazil. Thesis

(M.C.P.) Massachusetts Institute of Technology, Dept. of Urban Studies and
Planning. Fulltext available at
Gutarra, M.L.E., Godoy, M.G., Maugeri, F., Rodrigues, M.I., Freire, D.M.G. & Castilho, L.R.
(2009). Production of an acidic and thermostable lipase of the mesophilic fungus
Penicillium simplicissimum by solid-state. Bioresource Technology. 100: 5249-5254.
Ha, S.H., Lan, M.N., Lee, S.H., Hwang, S.M. & Koo, Y-M. (2007). Lipase-catalyzed biodiesel
production from soybean oil in ionic liquids. Enzyme and Microbial Technology. 41(4):
480-483.
Haas, M.J. & Foglia, T.A. (2003). In situ production of fatty acid alkyl esters. United States
patent and trademark office pre-grant publication, Dec 2003 patno: US20030229237
Hara, A. & Radin, N.S. (1978). Lipid extraction of tissues with a low-toxicity solvent.
Analytical Biochemistry. 90: 420.
Harun, R., Singh, M., Forde, G.M. & Danquah, M.K. (2010). Bioprocess engineering of
microalgae to produce a variety of consumer products. Renewable and Sustainable
Energy Reviews. 14: 1037-1047.
Helwani, Z., Othman, M.R., Aziz, N., Fernando, W.J.N. & Kim, J. (2009). Technologies for
production of biodiesel focusing on green catalytic techniques: A review. Fuel
Processing Technology.
90: 1502.